The recent characterization of prion proteins has revealed that a primary sequence of amino acids can give rise to at least two distinctly folded conformers. Surprisingly, the prion conformer can template the conversion of fully folded protein into the prion form. Thus, prion proteins provide an example of dynamic protein conformations and reveal that proteins can convert from one stable conformer to another. The experiments described herein are designed to monitor the interplay between conformational states in vivo. The information obtained from these experiments will further our understanding of structural plasticity and provide targets for therapeutic intervention directed at perturbing and/or reversing the equilibrium between conformations in diseases associated with abnormal protein folding. In recent years, Saccharomyces cerevisiae has emerged as an experimentally tractable model system for the study of self-replicating changes in protein conformation in vivo. One particular prion determinant in yeast, Sup35, normally functions to terminate translation. However, in [PSI+] cells, Sup35 forms insoluble aggregates. Despite the fact that Sup35's activity in translation termination is required for cell viability, [PSI+] cells survive even though nearly all of the Sup35 is aggregated at steady-state. One can only reconcile the viability of [PSI+] cells with the existence of a soluble pool of active Sup35 in addition to the aggregated form in these cells. How do these distinct pools arise and persist? Our hypothesis is that the functional pool of Sup35 originates in part from the recycling of proteins from aggregates. This hypothesis is based on several observations. First amyloids can exchange with the soluble pool of protein in vitro. Second, maintenance of the prion state requires the molecular disaggregase Hsp104 and third, the Hsp104 homolog in E. coli, CIpB, remodels aggregates into functionally competent conformers. While these observations suggest that aggregates are in dynamic exchange with the soluble conformer, there is no direct evidence for exchange in vivo. Also, aggregate recycling contradicts the current view that the prion conformer is stable and recalcitrant to normal cellular quality control pathways. Thus, the overall objective of this proposal is to elucidate the relationship between distinct conformations of a single prion protein in the same cell by linking Sup35 activity in [PSI+] cells to either newly made or existing protein. The specific aims are to: 1) Establish assays to monitor the activity of Sup35 and 2) Link the functional pool of Sup35 to newly synthesized and/or recycled Sup35 through modulation of the Sup35 prion cycle. [unreadable] [unreadable] [unreadable]